Humanization of an anti-CεmX monoclonal antibody 4B12 for potential clinical use in targeting mIgE⁺B cells (140.19)

Abstract

The CεmX domain of 52 a.a. residues, located between the CH4 domain and membrane-anchor segment of human membrane-bound ? chain on mIgE+ B cells, is a unique antigenic site for the immunological targeting of those B cells. We have demonstrated that a chimeric form of anti-CεmX monoclonal antibody (mAb), 4B12, can induce apoptosis and ADCC against a human mIgE.Fc+ B cell transfectoma via effector cells from PBMC in vitro (presented by another abstract in this meeting). To enhance the clinical applicability of 4B12, we have humanized it by first grafting its six complementary-determining regions (CDRs) into human Ig VH subgroup IV and V? subgroup II frameworks (FWs). Those residues in the FWs of VH and VL of the parental murine 4B12 that are essential for Fv dimer interaction and CDR conformation were identified by analyzing the binding affinity of a number of designed Ab variants with site-directed mutations, using competitive ELISA and surface plasmon resonance analysis. A humanized 4B12 retaining the affinity of its parent was obtained by adopting all FWs of the selected human V? template without any change and all FWs of the selected VH with only one residue change. The Val at position 71 of the human VH was replaced by Arg at the corresponding position of the murine VH. Based on molecular modeling analysis, it is likely that such a change is probably crucial for maintaining the proper structural packing of Arg71 with Ile29 in the CDRH1 and Gly55 in the CDRH2. The KD of the chimeric and humanized 4B12 are 5.37×10-9 M-1 and 5.76×10-9 M-1, respectively. Affinity enhancement of humanized 4B12 by selective CDR mutations is in progress.